INVESTIGATING THE ROLE OF EZH1 MEDIATED H3K27ME3 IN MOUSE CORTICAL NEURONS

Histone 3 lysine 27 trimethylation (H3K27me3) is a repressive histone modification enriched on silenced genes. Deposition of H3K27me3 is catalysed by Polycomb Repressive Complex 2 (PRC2), which contains either Enhancer of Zeste Homolog 1 (EZH1) or EZH2 as its catalytic subunit. While EZH2 predominantly mediates H3K27 methylation in proliferative cells, EZH1 is the main PRC2 methyltransferase in postmitotic neurons (unpublished data). However, the role of EZH1-mediated H3K27me3 during later stages of neuronal development and mature cerebral cortex remains poorly understood. To address this, we investigate the function of EZH1 in the mouse cerebral cortex using Ezh1 conditional knockout mice crossed with the Neurod1-Cre line. In this model, Cre expression is initiated in postmitotic neurons. Preliminary single-nucleus RNA sequencing revealed neuron-type specific transcriptional changes upon loss of EZH1-mediated H3K27me3, with layer 2/3 and layer 4/5 intratelencephalic (IT) neurons showing the strongest effects. To validate and extend these findings, we will isolate these neuronal populations using fluorescence-activated nuclei sorting (FANS). We will first determine the timing of H3K27me3 loss in these cortical neurons and assess associated gene expression changes by RNA sequencing. In parallel, we will characterise EZH1-dependent chromatin regulation using CUT&RUN to map H3K27me3 localisation and PLAC-seq to map chromatin interactions across distinct neuronal subtypes. Finally, we will examine cortical morphology by immunohistochemistry to assess potential structural and cellular consequences of EZH1 loss. Together, this work will unravel the role of EZH1-mediated H3K27me3 in cortical neurons to understand basic mechanisms of epigenetic regulation.